Methods of using cell-cycle inhibitors to modulate one or more properties of a cell culture

Abstract

Methods of modulating the properties of a cell culture expressing a protein of interest are provided. In various embodiments the methods relate to the addition of cell cycle inhibitors to growing cell cultures.

1 . A method of increasing specific productivity in a mammalian cell culture expressing a recombinant protein comprising
 establishing a mammalian cell culture in a culture medium;   inducing cell growth-arrest by contacting the cell culture with a culture medium comprising a cell cycle inhibitor that has an IC 50  in a CDK4 enzyme inhibition assay of less than about 20 nM; and   maintaining the cell culture in a growth-arrested state by contacting the culture with a culture medium comprising a cell cycle inhibitor.   
     
     
         2 . A method of increasing recombinant protein production in a mammalian cell culture expressing a recombinant protein comprising
 establishing a mammalian cell culture in a culture medium;   inducing cell growth-arrest by contacting the cell culture with a culture medium comprising a cell cycle inhibitor that has an IC 50  in a CDK4 enzyme inhibition assay of less than about 20 nM; and   maintaining the cell culture in a growth-arrested state by contacting the culture with a culture medium comprising a cell cycle inhibitor.   
     
     
         3 . A method of limiting a mammalian cell culture expressing a recombinant protein at a desired packed cell volume comprising
 establishing a mammalian cell culture in a culture medium;   inducing cell growth-arrest by contacting the cell culture with a culture medium comprising a cell cycle inhibitor that has an IC 50  in a CDK4 enzyme inhibition assay of less than about 20 nM; and   maintaining the cell culture in a growth-arrested state by contacting the culture with a culture medium comprising a cell cycle inhibitor.   
     
     
         4 . The method of  claim 1 , wherein the cell culture is contacted with culture medium comprising a cell cycle inhibitor on or before day 3 of the culture. 
     
     
         5 . The method  claim 1 , wherein induction of cell growth-arrest takes place prior to a production phase. 
     
     
         6 . The method  claim 1 , wherein induction of cell growth-arrest takes place during a production phase. 
     
     
         7 . The method of  claim 1 , wherein the cell cycle inhibitor has an IC 50  in a CDK4 cell assay of less than about 100 nM. 
     
     
         8 . The method of  claim 1 , wherein the cell cycle inhibitor is selected from the group consisting of: N-(6-(4-(dimethylamino)-1-piperidinyl)-3-pyridazinyl)-9-(trans-4-methylcyclohexyl)-9H-pyrido[4′,3′,:4,5]pyrrolo[2,3-d]pyrimidin-2-amine, 9-cyclopentyl-N-(6-(4-(dimethylamino)piperidin-1-yl)pyridazin-3-yl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine, 9-(4-methylcyclohexyl)-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine, 8-(6-((9-(4-methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)pyridazin-3-0)-2-thia-8-azaspiro[4.5]decane 2,2-dioxide, N-(6-(4-(dimethylamino)piperidin-1-yl)pyridin-3-0)-4,4-dimethylspiro[cyclohex[2]ene-1,9′-pyrido[4′,3′:3,4]cyclopenta[1,2-d]pyrimidin]-2′-amine 
       and the salts, esters, and amides, thereof. 
     
     
         9 .- 12 . (canceled) 
     
     
         13 . The method  claim 1 , wherein the cell culture is grown by a method selected from the group consisting of batch culture, fed-batch culture, perfusion culture, and combinations thereof. 
     
     
         14 . The method  claim 1 , further comprising a temperature shift from 36° C. to 31° C. 
     
     
         15 . The method of  claim 1 , wherein the method further comprises limitation of a key nutrient in the culture medium. 
     
     
         16 .- 20 . (canceled) 
     
     
         21 . The method  claim 1 , wherein the concentration of cell cycle inhibitor in the culture medium is between 0.5 and 5 mM. 
     
     
         22 . The method  claim 1 , wherein the concentration of cell cycle inhibitor in the culture medium is between 1 and 4.0 mM. 
     
     
         23 . The method  claim 1 , wherein the concentration of cell cycle inhibitor in the culture medium is between 2 and 3.0 mM. 
     
     
         24 . The method  claim 1 , wherein the concentration of cell cycle inhibitor in the culture medium is between 2.5 and 5 mM. 
     
     
         25 . The method  claim 1 , wherein the concentration of cell cycle inhibitor in the culture medium is between 3.5 and 5 mM. 
     
     
         26 . The method of  claim 1 , wherein the packed cell volume during a production phase is less than or equal to 35% 
     
     
         27 . The method of  claim 1 , wherein the packed cell volume is less than or equal to 30%. 
     
     
         28 . The method of  claim 1 , wherein the viable cell density of the mammalian cell culture at a packed cell volume less than or equal to 35% is 10×10 6  viable cells/ml to 80×10 6  viable cells/ml. 
     
     
         29 . The method  claim 1 , wherein the viable cell density of the mammalian cell culture is 20×10 6  viable cells/ml to 30×10 6  viable cells/ml. 
     
     
         30 .- 44 . (canceled) 
     
     
         45 . The method of  claim 1 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         46 . The method of  claim 1 , wherein the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine. 
     
     
         47 . The method of  claim 46 , further comprising a step of harvesting the recombinant protein produced by the cell culture. 
     
     
         48 . The method of  claim 47 , wherein the recombinant protein produced by the cell culture is purified and formulated in a pharmaceutically acceptable formulation. 
     
     
         49 . (canceled)